A Trojan horse mechanism of bacterial pathogenesis against nematodes
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Fatal attraction: Bacterial bait lures worms to their death Kendra P. Rumbaugh 1 Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX 79430 D uring the past decade, the roundworm Caenorhabditis ele- gans has become a popular model for the study of host/ pathogen relationships, leading to a wealth of information about microbial virulence factors and host defense path- ways (1). Although the complicated inter- actions between C. elegans and the many pathogens that it encounters in the soil have become clearer in recent years, there is still much to learn. What are the cues that worms use to detect food sources? How do worms choose which bacterial species to eat and which to leave alone? Once a pathogen is encountered, what are the microbial killing mechanisms and the nematode’s survival mechanisms? The paper by Niu et al. (2) in PNAS provides a rare 360° view of one C. elegans/patho- gen relationship. It describes the Bacillus nematocida signals that attract C. elegans, the virulence factors it uses to kill worms from within, and the specific host proteins targeted. Smells Good Enough to Eat Roundworms burrowing through the soil encounter thousands of species of bacte- ria, but how do they find safe food choices on this vast buffet? A major part of the nematode’s ability to distinguish between food sources relies on a sophisticated chemosensory system that enables it to sense and respond to a wide range of volatile and water-soluble chemicals (3). This network of 32 chemosensory neurons, located at both the anterior and the pos- terior ends of the nematode (amphid, phasmid, and inner labial neurons in Fig. 1), facilitates olfactory chemotaxis, allow- ing worms to move toward or away from odors associated with food. More than 5% of C. elegans genes and ≈1,000 G-protein– coupled chemoreceptors mediate odor perception, hinting at the magnitude of its importance (4). Bargmann’s group and others have identified a wide array of chemicals that either attract or repel C. elegans (3), and it has long been ob- served that C. elegans displays distinct preferences in food choice (5–7); however, the specific identity of the attractants and repellants responsible for chemotaxis in the worm’s natural environment has re- mained largely unknown. C. elegans is attracted and repelled by some amino acids and bacterial metabo- lites, which in theory could serve as food choice odorants (3), and a few recent studies have matched specific attractants and/or repellants to the microbial species producing them. For example, after ob- serving that quorum sensing (QS) strains of Pseudomonas aeruginosa were more at- tractive to C. elegans than QS-negative strains, Beale et al. (8) demonstrated that worms were attracted to the acylated ho- moserine lactones that serve as inter- bacterial chemical signals that facilitate QS in Pseudomonas and other Gram- negative bacteria. Pradel et al. (9) later reported that the cyclic lip- odepsipentapeptide, serrawettin W2, was a C. elegans repellant produced by Serratia marcescens. In PNAS, Niu et al. (2) iden- tify several volatile organic compounds (VOCs) as attractants for B. nematocida and Escherichia coli. Niu et al. (2) use a simple migration assay to demonstrate the intense attraction of C. elegans to B. nematocida. When a lawn of B. nematocida was placed on an inverted agar layer in the upper lid of a petri plate, 56% of the nematodes mi- grated toward the bacterial lawn by labo- riously climbing up the walls of the petri dishes. Because only 1.8% and 12% of worms migrated to agar alone or to a lawn of E. coli (the worm’s normal dietary spe- cies in the laboratory), respectively, the authors speculated that B. nematocida produced one or more volatile compounds that attracted C. elegans. Using solid-phase microextraction–gas chromatographic– mass spectrometric analysis, Niu et al. (2) identify 17 distinct VOCs from cultures of B. nematocida, and of the several com- pounds that were tested individually as odorants, benzyl benzoate, benzaldehyde, 2-heptanone, and acetophenone, each showed potent nematode-attracting abili- ties. Although benzaldehyde and 2-hepta- none have previously been reported as attractants for C. elegans (3), this report identifies them as attractants produced by a specific bacterial species and provides more evidence that pathogenic microbes can use this olfactory chemotaxis to attract nematodes. Interestingly, benzyl benzoate was previously reported to repel C. elegans (3). Given that many chemicals can serve as odorants at one concentration and re- pellants at others, it will be important to ascertain the concentration of odorants expected to be produced in situ. Bacteria Bite Back One could reason that producing a nema- tode repellant would be advantageous to bacteria in the soil, but could the chemo- attractive nature of these microbial prod- ucts actually benefit bacteria as well? There are dozens of bacterial species pathogenic to C. elegans that kill worms by a variety of mechanisms. Typically, pathogens that are consumed by worms accumulate in the intestine and kill either by a persistent infection, which causes significant intestinal distension and death over a period of days, or by a diffusible toxin-mediated “fast-killing” (reviewed in ref. 10). Less frequently, some pathogens kill by direct invasion from the outside. For example, Brevibacillus laterosporus and some pathogenic fungi secrete proteases that degrade the worm’s cuticle and cause an invasive, disseminated lethal infection (10, 11). Previously, there had been no reports of ingested pathogens that invade intestinal cells from the digestive tract, which suggested that C. elegans intesti- nal cells were remarkably resistant to Fig. 1. C. elegans uses a sophisticated chemosensory system to sense and move toward microbial by- products in its environment. Once a pathogen is consumed, it may kill worms slowly by dividing within the worm’s intestine and causing a prolonged, possibly disseminated infection or kill them quickly by releasing diffusible toxins. However, in PNAS, Niu et al. (2) describe a virulence strategy by which B. nematocida attracts C. elegans with a variety of volatile organic compounds and, once consumed, kills the worm by excreting two proteases, Bace16 and Bae16, which target host proteins essential for in- testinal homeostasis. Author contributions: K.P.R. wrote the paper. The author declares no conflict of interest. See companion article on page 16631. 1 E-mail: [email protected]. www.pnas.org/cgi/doi/10.1073/pnas.1011935107 PNAS | September 21, 2010 | vol. 107 | no. 38 | 16411–16412 COMMENTARY
Transcript of A Trojan horse mechanism of bacterial pathogenesis against nematodes
Fatal attraction: Bacterial bait lures worms to their deathKendra P. Rumbaugh1
Department of Surgery, Texas Tech University Health Sciences Center, Lubbock, TX 79430
During the past decade, theroundworm Caenorhabditis ele-gans has become a popularmodel for the study of host/
pathogen relationships, leading toa wealth of information about microbialvirulence factors and host defense path-ways (1). Although the complicated inter-actions between C. elegans and the manypathogens that it encounters in the soilhave become clearer in recent years, thereis still much to learn. What are the cuesthat worms use to detect food sources?How do worms choose which bacterialspecies to eat and which to leave alone?Once a pathogen is encountered, what arethe microbial killing mechanisms and thenematode’s survival mechanisms? Thepaper by Niu et al. (2) in PNAS providesa rare 360° view of one C. elegans/patho-gen relationship. It describes the Bacillusnematocida signals that attract C. elegans,the virulence factors it uses to kill wormsfrom within, and the specific hostproteins targeted.
Smells Good Enough to EatRoundworms burrowing through the soilencounter thousands of species of bacte-ria, but how do they find safe food choiceson this vast buffet? A major part of thenematode’s ability to distinguish betweenfood sources relies on a sophisticatedchemosensory system that enables it tosense and respond to a wide range ofvolatile and water-soluble chemicals (3).This network of 32 chemosensory neurons,located at both the anterior and the pos-terior ends of the nematode (amphid,phasmid, and inner labial neurons in Fig.1), facilitates olfactory chemotaxis, allow-ing worms to move toward or away fromodors associated with food. More than 5%of C. elegans genes and ≈1,000 G-protein–coupled chemoreceptors mediate odorperception, hinting at the magnitude of itsimportance (4). Bargmann’s group andothers have identified a wide array ofchemicals that either attract or repelC. elegans (3), and it has long been ob-served that C. elegans displays distinctpreferences in food choice (5–7); however,the specific identity of the attractants andrepellants responsible for chemotaxis inthe worm’s natural environment has re-mained largely unknown.C. elegans is attracted and repelled by
some amino acids and bacterial metabo-lites, which in theory could serve as foodchoice odorants (3), and a few recentstudies have matched specific attractants
and/or repellants to the microbial speciesproducing them. For example, after ob-serving that quorum sensing (QS) strainsof Pseudomonas aeruginosa were more at-tractive to C. elegans than QS-negativestrains, Beale et al. (8) demonstrated thatworms were attracted to the acylated ho-moserine lactones that serve as inter-bacterial chemical signals that facilitateQS in Pseudomonas and other Gram-negative bacteria. Pradel et al. (9) laterreported that the cyclic lip-odepsipentapeptide, serrawettin W2, wasa C. elegans repellant produced by Serratiamarcescens. In PNAS, Niu et al. (2) iden-tify several volatile organic compounds(VOCs) as attractants for B. nematocidaand Escherichia coli.Niu et al. (2) use a simple migration
assay to demonstrate the intense attractionof C. elegans to B. nematocida. Whena lawn of B. nematocida was placed on aninverted agar layer in the upper lid ofa petri plate, 56% of the nematodes mi-grated toward the bacterial lawn by labo-riously climbing up the walls of the petridishes. Because only 1.8% and 12% ofworms migrated to agar alone or to a lawnof E. coli (the worm’s normal dietary spe-cies in the laboratory), respectively, theauthors speculated that B. nematocidaproduced one or more volatile compoundsthat attracted C. elegans. Using solid-phasemicroextraction–gas chromatographic–mass spectrometric analysis, Niu et al. (2)identify 17 distinct VOCs from cultures ofB. nematocida, and of the several com-pounds that were tested individually asodorants, benzyl benzoate, benzaldehyde,2-heptanone, and acetophenone, eachshowed potent nematode-attracting abili-ties. Although benzaldehyde and 2-hepta-none have previously been reported asattractants for C. elegans (3), this report
identifies them as attractants produced bya specific bacterial species and providesmore evidence that pathogenic microbescan use this olfactory chemotaxis to attractnematodes. Interestingly, benzyl benzoatewas previously reported to repel C. elegans(3). Given that many chemicals can serveas odorants at one concentration and re-pellants at others, it will be important toascertain the concentration of odorantsexpected to be produced in situ.
Bacteria Bite BackOne could reason that producing a nema-tode repellant would be advantageous tobacteria in the soil, but could the chemo-attractive nature of these microbial prod-ucts actually benefit bacteria as well?There are dozens of bacterial speciespathogenic to C. elegans that kill wormsby a variety of mechanisms. Typically,pathogens that are consumed by wormsaccumulate in the intestine and kill eitherby a persistent infection, which causessignificant intestinal distension and deathover a period of days, or by a diffusibletoxin-mediated “fast-killing” (reviewed inref. 10). Less frequently, some pathogenskill by direct invasion from the outside.For example, Brevibacillus laterosporus andsome pathogenic fungi secrete proteasesthat degrade the worm’s cuticle and causean invasive, disseminated lethal infection(10, 11). Previously, there had been noreports of ingested pathogens that invadeintestinal cells from the digestive tract,which suggested that C. elegans intesti-nal cells were remarkably resistant to
Fig. 1. C. elegans uses a sophisticated chemosensory system to sense and move toward microbial by-products in its environment. Once a pathogen is consumed, it may kill worms slowly by dividing withinthe worm’s intestine and causing a prolonged, possibly disseminated infection or kill them quickly byreleasing diffusible toxins. However, in PNAS, Niu et al. (2) describe a virulence strategy by which B.nematocida attracts C. elegans with a variety of volatile organic compounds and, once consumed, killsthe worm by excreting two proteases, Bace16 and Bae16, which target host proteins essential for in-testinal homeostasis.
Author contributions: K.P.R. wrote the paper.
The author declares no conflict of interest.
See companion article on page 16631.1E-mail: [email protected].
www.pnas.org/cgi/doi/10.1073/pnas.1011935107 PNAS | September 21, 2010 | vol. 107 | no. 38 | 16411–16412
COMMENTARY
microbial invasion. However, in PNAS,Niu et al. (2) describe two proteases pro-duced by B. nematocida that target theintestine from within.Niu et al. (2) find that the culture fil-
trates from B. nematocida strains in-capable of producing the serine andneutral proteases, Bace16 and Bae16, re-spectively, displayed significantly lowerlevels of proteolytic and nematocidal ac-tivities. Whereas 90% of worms were aliveafter a 5-d exposure to E. coli culture fil-trates, only 5% were alive after a 5-d ex-posure to culture filtrate from wild-type B.nematocida. However, 80% of nematodeswere still viable after exposure to the fil-trates of a double bace16/bae16 knockoutstrain. Localization experiments withfluorescence-tagged proteins demon-strated that the two proteases localizedmainly to the intestine of nematodes andcorrelated with severe intestinal damage,including disordered and loose intestinalwalls and destabilized microvilli along thebrush border.Previously, the major proposed mecha-
nism for protease-mediated microbialpathogenesis has been via the breakdownof the nematode cuticle. Thus, Niu et al.(2) tested whether crude protease extractsfrom wild-type B. nematocida or bace16/bae16 knockouts would cause nematodemortality either when microinjected intothe intestine or when applied to their cu-ticle. In support of their localization ex-
periments, Niu et al. (2) observe that C.elegans receiving intestinal microinjectionsof protease extracts from wild type, but notfrom the mutant strains, displayed signifi-cantly higher levels of mortality than thosetreated externally with proteases. Niu et al.(2) next use high-resolution, 2D gel elec-trophoresis to identify the host proteins
Niu et al. propose that
B. nematocida actively
lures in C. elegans by
producing odorants.
within the nematode epithelia targetedby B. nematocida proteases. Twelve nem-atode proteins were deemed as preferen-tial targets because their expressiondecreased more than threefold after 1 hof treatment with Bace16/Bae16. Severalof the identified proteins, includingPEPCK and VHA-8, are essential forintestinal function and support the con-clusion that B. nematocida excretes pro-teases that act on the nematode intestineand represents a unique mechanism of pa-thogenesis.
Finicky EaterNiu et al. (2) propose that B. nematocidaactively lures in C. elegans by producing
odorants that serve as attractants andthat, once inside the intestine, usesa “Trojan horse” mechanism to kill theworms. This implies that these encountersare not simply random, but that specificpredatory mechanisms have evolved inbacteria. This is an interesting conceptthat undoubtedly needs further in-vestigation because if microbes are thetrue predators, is C. elegans completelydefenseless to their baits and lures? Itturns out that the worm is not as dumb asit looks. In fact, C. elegans can use olfac-tory learning to recognize the odorantsproduced by some pathogens as cues toavoid them (6, 8). Thus, odors that servedas attractants to naive worms may be-come repellants after the worms are ex-posed to the pathogens excreting them.Furthermore, because many of the bac-terial metabolites that C. elegans findsattractive are produced by friends andfoes alike, these olfactory learningmechanisms must be highly specific butadaptable. It remains to be seen whetherC. elegans can learn to avoid the VOCsproduced by B. nematocida after it hasbeen exposed to the pathogen and, if so,whether this negative conditioning willcause the worm to avoid all microbes se-creting similar VOCs.
ACKNOWLEDGMENTS. Research in the RumbaughLaboratory is supported by the AmericanDiabetes Association.
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16412 | www.pnas.org/cgi/doi/10.1073/pnas.1011935107 Rumbaugh
